The overall objective of this project is to understand the mechanism of RNA synthesis in mouse hepatitis virus (MHV), a member of coronaviruses. This proposal is based on our recent observations that MHV synthesizes seven intracellular virus-specific RNAs which overlap sequences at the 3'-ends and also share at least terminal five nucleotides. These observations suggest a complex mechanism for the synthesis of MHV RNAs. In this project, we plan to understand this mechanism by the following approaches: (1) Determine the 5'-end sequences of MHV mRNAs by direct RNA sequencing to determine how many nucleotides are shared at 5'-ends. (2) Perform molecular cloning of the subgenomic and virion genomic RNA. These cloned DNAs will be used for S1 mapping and sequence determination to detect RNA splicing, modifications or any other forms of processing. The DNA sequences will also be used for determinations of the translational capacity of the mRNAs. (3) Isolate and characterize the double-stranded (ds) RNA replicative form to determine the number of ds RNA species. The corresponding negative-stranded RNA will also be detected by "Northern" blotting techniques. (4) The RNA products of the in vitro RNA polymerase reactions will also be characterized. These products will represent the primary transcriptional products, possibly without extensive processing. These products will be compared with the RNAs isolated from the MHV-infected cells. These studies should be able to yield definitive informations about the mechanism of MHV RNA synthesis.